PROJECT SUMMARY/ABSTRACT Disruption of gene expression regulation is thought to underlie up to 90% of congenital heart defects (CHD) and is a major contributor to adult-onset complex cardiovascular diseases (CVD). A major source of this disruption is germline DNA mutations that alter transcription factor binding sites in cis-regulatory elements such as enhancers. Although we have tools available to identify these mutations in a patient, we cannot reliably predict which genes? expression patterns may be affected by the mutation because enhancers are often located far from the genes they regulate. Through long-range DNA looping interactions that place an enhancer element in physical proximity with the target gene?s promoter, transcription factors bound to the enhancer are able to activate gene expression. Therefore, it is critical that we associate enhancer elements to their target genes in the context of heart development and understand the extent to which DNA mutations impair enhancer function. Aim 1 of this research proposal will utilize the genome-wide chromosome conformation capture method, promoter-capture Hi-C, to identify all long-range promoter interactions in induced pluripotent stem cells (iPSCs), mesoderm precursors, and iPSC-derived cardiomyocytes. Integration of chromatin data for active enhancers will then enable identification and assignment of each enhancer to its target gene. Comparison of gene regulatory profiles during the course of the differentiation will enhance understanding of the regulatory logic underlying heart development and provide a map of DNA regions that, if harboring a mutation, may contribute to developmental heart defects and disease. Aim 2 of this research proposal involves the development of a novel metric to assess whether a distal DNA mutation alters gene expression. Promoter-capture Hi-C will be conducted in iPSC-derived cardiomyocytes for ten genetically distinct individuals. Extensive genotyping information for these individuals will then be used to assess the contribution of genetic variants to enhancer-promoter interaction strength. This information will enable us to better classify DNA mutations as potential modifiers of gene expression and has the advantage of not relying on gene expression measurements. Overall, these studies will provide a frame- work within which we can analyze genetic mutations and predict their effect on gene regulation in the context of both heart development and complex cardiovascular disease.